Septic shock is the common endpoint of uncontrolled infection, with 750,000 cases and $17 billion spent each year in the United States alone. During Gram negative sepsis, LPS hyperactivates innate immune sensors, driving the systemic inflammation and vascular collapse seen in septic shock. Beginning 17 years ago, TLR4 was shown to sense extracellular and vacuolar LPS, and to contribute to septic shock. Nevertheless, despite intensive research into the immune dysfunction underlying TLR4 activation and septic shock, new treatments have repeatedly failed to show clinical efficacy. Using Burkholderia thailandensis, we have now shown that LPS is sensed in the cytosol through caspase- 11, the noncanonical inflammasome. Indeed, Dr. Shao demonstrated that the lipid A portion of LPS is directly detected by interaction with the CARD domain of murine caspase-11, and its human homologues caspases-4 and -5. We further showed that caspase-11 is essential for host defense, including B. thailandensis and B. pseudomallei - the latter of which is a potential biologic weapon and etiologic agent of melioidosis. Nevertheless, while many advances have been made, many mechanisms within this novel cytosolic LPS surveillance system remain poorly characterized, including the importance of priming upstream of caspase-11/4/5, the structural features of lipid A that are detected, and the clearance mechanisms engaged downstream of the caspases. Without establishing the normal operation of the noncanonical inflammasome, it will be difficult to understand the pathological hyperactivation of this pathway during sepsis. In AIM 1, we propose to study the structural features of lipid A that are required for detection by caspase- 11, -4, and -5. Structures that bind to the caspase, but fail to activateit are potential competitive inhibitors for the treatment of septic shock. Other structures that activate caspase-4/5 but show minimal TLR4 activity could be novel adjuvants. In AIM 2 we use B. thailandensis as an in vivo probe to explore the potential effector mechanisms downstream of cytosolic LPS detection that enable resistance to cytosolic infection. Many mechanisms have been advanced based on in vitro work, but none have been thoroughly explored in vivo. We investigate the hypothesis that pyroptosis is the primary effector mechanism of caspase-11. If valid, this insight would promote sepsis therapies aimed at ameliorating the consequences of large-scale cellular pyroptotic lysis. Finally, in AIM 3 we explore caspase-4 and -5 in vivo, using mice that are humanized for the cytosolic LPS surveillance pathway. These results will determine how these two caspases are similar and dissimilar to their murine homolog caspase-11, and will instruct the translational relevance of murine studies with respect to human disease.